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Transdifferentiation induced by gene transfer
seminars in
CELL BIOLOGY, Vol 6, 1995: pp 157–163
Transdifferentiation induced by gene transfer Petra Boukamp
and which by itself is able to induce transdifferentiation even across developmental cell lineage borders.
While for many tissues the differentiation process is well characterized, little is known about ‘master switch’ genes determining a specific differentiation pathway and having the potential to induce this process in a cell designed for a different differentiation pathway. Based on heterokaryon and 5-aza-cytidine-induced hypomethylation experiments, the muscle determination gene MyoD1 was identified and isolated, which was shown to induce myogenic differentiation even in cells of ectodermal lineage. Since transdifferentiation studies could also be performed in drosophila in vivo by ‘false’ expression of developmental genes, it is tempting to speculate that experimentally induced transdifferentiation mimics processes during embryonic development and tissue maturation.
Transdifferentiation by somatic cell fusion First evidence for the existence of transacting factors able to either repress or induce the expression of differentiated functions derived from somatic cell hybrid studies in the early 70s, when two cell types of different origin were fused with each other (see ref 1). However, due to frequent loss of chromosomes in these somatic cell hybrids, they proved to be rather unstable and highly variable model systems. Based on this, Blau and coworkers developed a heterokaryon system in which the intact nuclei of both fused cell types were stably maintained for the length of the experiments. Using representatives of all three embryonic lineages, heterokaryon experiments for the first time allowed to address the question as to what extent the origin of one fusion partner would influence the expression of the other along a different differentiation pathway. By fusion of mouse muscle cells with human amniotic fibroblasts, it was possible to show that a set of human muscle genes was activated including those coding for proteins of the contractile apparatus, membrane components and muscle specific enzymes.1,2 Furthermore, these genes were not induced randomly but followed the timecourse of human myogenesis.3 In this context, it is interesting to note that muscle gene activation was generally seen when primary (normal) human nonmuscle cells were fused with the mouse muscle cells, although the extent varied and muscle genes were expressed more rapidly and to a higher degree in more closely related cell types (fibroblasts [mesoderm] versus keratinocytes [ectoderm] or hepatocytes [endoderm]. Transformed aneuploid cells, on the other hand, often failed to express muscle markers. These primary findings were not only substantiated by others using similar cell types, but could also be extended to heterokaryons, where adult mouse erythroleukemia cells were fused with either human fetal erythroid or other non-erythroid cells and monitored for activation of the adult form of the globin gene.4
Key words: somatic-cell-fusion / 5-aza cytidine / epidermal differentiation / MyoD1-transfection
TOTIPOTENT CELLS DEVELOP into three distinct cell lineages in the very early embryo — ectoderm, endoderm and mesoderm. Once a cell is determined, this cell type is bound to specialize along a specific differentiation pathway, i.e. to develop into tissues such as muscle or epidermis and this determination is stably inherited by all daughter cells. From the characterization of the different differentiation pathways we have learned a lot about structural genes, membrane components involved in cell–cell and cell–matrix interactions, or enzymes that are specifically expressed and are characteristic for a given tissue type. These studies, however, did not lead to the identification of genes, so called determination genes, which control the concerted action of a differentiation-specific pathway. The first and at present still only known tissue determinator is the myogenic determination gene MyoD1, which was identified through ‘transdifferentiation’ studies where cells developed into an unrelated phenotype by ectopically forced expression of otherwise silent genes
From the German Cancer Research Center, Department of Carcinogenesis and Differentiation, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany ©1995 Academic Press Ltd 1043-4682/95/030157 + 07$8.00/0
157
P. Boukamp sional gel-electrophoresis studies indicated that only a few specific genes could be involved in lineage determination and that regulatory loci responsible for the myogenic determination, rather than differentiation-specific structural genes, were activated by 5-azaCR.8 Similarly, in mouse teratocarcinoma-derived fibroblasts, hypomethylation by 5-aza-CR resulted in a transient expression of a 55 kd keratin protein (K14), an intermediate filament protein characteristic for epithelial cells and generally absent in fibroblasts. From these cells, clones with epithelial morphology could be established, which not only expressed this keratin but, in addition, the whole set of epidermal keratins and desmosomes. Furthermore, these cells were able to differentiate into corneocytes.9,10 Thus, several authors were able to show that by treating cells with 5-aza-CR, genes were activated which then induced an alternative differentiation pathway; i.e. induced transdifferentiation. Moreover, since as an intriguing feature this cytidine analog selectively activates genes instead of causing genomewide random derepression this approach closely resembles the experimental design of transfer of specific genes. Thus, it was not surprising that the 5-aza-CR treated C3H 10T1/2 clone 8 cells were further used as a model system to determine genetic factors controlling the different mesodermal cell lineage determination and differentiation.
Thus, it was suggested from these heterokaryon experiments (i) that alternative differentiation pathways were not irreversibly blocked in already determined somatic cells and (ii) since myogenesis could be induced in the nucleus of the fusion partner this alternative differentiation pathway was not silenced by inactivating all the individual differentiation markers but only one or a few transacting factors.
Transdifferentiation by 5-azacytidine treatment This first step of ‘transdifferentiation’ could be extended to an even more completely transdifferentiated phenotype from fibroblastic to myogenic by treating C3H 1OT1/2 cells with 5-azacytidine (5-azaCR). Although it does not represent a true gene transfer, these experiments were based on the activation of genes which under normal conditions were never expressed in these cells. Furthermore, it was only because of this experimental approach that the determination gene for myogenesis was identified and that the present gene transfer experiments became feasible. 5-aza-CR, originally developed as a cancer chemotherapeutic agent,5 is a cytidine analog carrying a nitrogen instead of a carbon atom at position 5 of the pyrimidine ring and is randomly incorporated into DNA. Since the azanucleoside ring cannot be methylated, this integration now leads to extensive hypomethylation in CpG residues via inhibition of DNA methylation.6 By treating C3H 10T1/2 clone 8 cells with 5-aza-CR these multipotent mesodermal cells could be induced to develop into three new mesenchymal phenotypes. They formed contractile striated muscle cells which expressed elevated levels of myosin ATPase activity and acetylcholine receptors, biochemically defined adipocytes which accumulated large lipid droplets, and chrondrocytes which were able to synthesize cartilage-specific proteins.7 These altered phenotypes were not due to selection of rare pre-existing subpopulations since a comparable conversion never occurred spontaneously, and they were shown to be inheritable by propagation of the cells in the absence of further drug treatment. From these studies it was suggested that regulation of the three differentiation pathways had been inactivated in the precursor cell but could be reactivated by hypomethylation of specific genes. It remained unclear from these early studies whether the whole set or only one or a few ‘master switch’ genes had to be activated by hypomethylation. Subsequent two-dimen-
Transdifferentiation by gene transfer Genomic DNA The hypothesis that the profound effects of 5-aza-CR on differentiation/transactivation was due to the activation of one or a few determination loci (‘master switch’ genes) was substantiated by exciting experiments performed by Lassar and coworkers.11 They were able to show that DNA from 5-aza-CR-derived myoblasts, when transfected into normal (untreated) C3H 10T1/2 cells, induced the emergence of myoblasts at a frequency expected when only a few demethylated loci were required.
The master regulatory gene for myogenesis MyoD1 In an attempt to identify the genes, Lassar and coworkers used the technique of subtractive cDNA hybridization using poly(A) + RNA from normal diploid mouse myogenic cells and the 5-aza-CR treated 158
Gene transfer 10T1/2 cells.12 By screening a myocyte cDNA library a number of cDNA clones could be isolated, one of which was called MyoD1 and was sufficient to convert 10T1/2 cells, and, to a lesser extent, mouse 3T3 fibroblasts and a variety of other cell lines into stable myoblasts upon transfection. Furthermore, MyoD1 could be shown to be normally expressed in mouse skeletal muscle in vivo and in myogenic cell lines in vitro but to be absent in other tested adult or newborn mouse tissues as well as in several non-differentiating myogenic cell lines.12 Concurrently, other MyoD-like genes were identified which could similarly convert 10T1/2 cells into myogenic lineages.13-15 Once the MyoD1 gene was isolated, a number of studies were performed to demonstrate its potential to convert different fibroblast, adipoblast and osteosarcoma cell lines16-18 into muscle cells. In addition, it was asked whether this single ‘master switch’ gene MyoD1 would be able to also convert cells from unrelated embryonic lineages to myogenesis, i.e. to express muscle markers. And indeed it could be shown that transfection of retinal pigmented cells, mouse melanoma cells and rat neuroblastoma cells with this single myogenic determination gene resulted in the formation of multinucleated myotubes and coexpression of the endogenous and myogenic differentiation program.19,20 We have performed an extensive study using cells of ectodermal lineage, the human skin keratinocyte line HaCaT.21 The rational for this study was that epidermal differentiation in its tissue-specific organization is extremely well defined and that experimental systems are available to study this complex system. Furthermore, the HaCaT cells, although immortal and aneuploid, had maintained a largely normal capacity for epidermal differentiation even during serial passaging, or when the cells became malignantly transformed, and remained capable to form an epidermis-like epithelium under physiological conditions in vivo (in transplants) which closely resembled that of normal human keratinocytes.22-24 Thus, the HaCaT cells provided an excellent model system to study short- and long-term effects following stable integration of the MyoD1 gene and further propagation of individual clones.25 As expected, a small fraction of HaCaT cells converted into multinucleated myotubes (Figure 1A) and expressed the typical structural proteins vimentin, desmin (Figure 1B) and myosin. However, when the cells were grown in transplants in vivo and were exposed to the influence of the connective tissue (known to be required for complete epidermal differentiation) in addition to its
Figure 1. (A) Culture of MyoD1 transfected HaCaT cells. On a background of typical epidermal cells multinucleated myotubes have formed which are stained with an antibody against myosin. (B) Immunofluorescence micrograph of a similar culture using an anti-desmin antibody. (C) Phase contrast micrograph of DTHMZ cells demonstrating a more fibroblastic morphology. Bar, 100µm
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P. Boukamp ng/ml; Boukamp and H¨ulsen, unpublished results). Furthermore, expression of growth factors characteristic for fibroblasts such as granulocyte- or granulocyte/macrophage-colony-stimulatory factor (G-CSF or GM-CSF) was similarly absent in HaCaT as in DTHMZ cells (Boukamp and M¨uller, unpublished results). Thus, these studies strongly suggest that there are independent regulatory pathways for (i) the expression of and reactivity to growth factors and (ii) ‘morphological’ differentiation. We further have to postulate that already for ‘morphological’ transdifferentiation from ectodermal to mesenchymal lineage one ‘master switch gene’ is not sufficient to overcome the stable intrinsic program (at least for epidermal differentiation), since variants similar to DTHMZ cells did not evolve from HaCaT or MyoD1- transfectants without 5-aza-CR treatment. Only by additional hypomethylation we obviously activated one or more of the genes required to induce this fully transdifferentiated phenotype.
intrinsic regulation, the myogenic differentiation pathway was down-regulated and the cells formed a normally structured and biochemically well-differentiated epidermis.25 (Figure 2A). From these MyoD1 transfectants we were able, in a step-wise fashion, to select for a variant with solely mesenchymal/myogenic characteristics. To potentiate the MyoD1 action, MyoD1-transfectants were treated with 5-aza-CR. Although this did not result in an increase in the number of myogenic cells in vitro, it clearly altered the response to intrinsic/extrinsic regulators of epidermal differentiation in vivo.25 The epithelium formed by these cells was morphologically unstructured and undifferentiated (Figure 2B). Nevertheless, differentiation markers were still expressed in a rather normal fashion. Since a similar dissociation of histo- and cytodifferentiation is seen during embryological development when the epidermis develops from a two- to a multi-layered tissue, the experimentally induced stage may represent the physiological ‘transition-phase’ and thus, the ModD1transfected, 5-aza-CR-treated HaCaT cells may provide a useful model to determine factors involved in epidermal maturation and differentiation. In these cultures a subpopulation (DTHMZ) evolved which, when selected on the basis of reduced adhesion, had reached a stage of ‘complete’ transdifferentiation25 (Figure 1C). Reduced adhesion could clearly be related to the loss of expression in a group of adhesion molecules, the integrins, which were transformed from a complex keratinocyte-like pattern to a more simple fibroblast-like one.26 As a functional consequence, growth on those matrices was poor where the respective receptors were missing. But even on growth-promoting substrates (fibronectin) the DTHMZ cells were no longer able to stratify when grown as transplants in vivo. They mostly remained as a monolayer (Figure 2C) and no longer expressed any of the epidermal differentiation markers either in vivo or in vitro. Instead, the number of cells expressing myogenic differentiation markers doubled and, additionally, other myogenic regulator genes such as myf-313 and myogenin shown to be activated in a positive autoregulatory loop with MyoD1,27 were seen. Despite of all these alterations, the functional switch to mesenchymal/myogenic phenotype was still not complete. Recent experiments have shown that TGF-β1, a potent growth-inhibitor for keratinocytes, which, however, stimulates growth of fibroblasts at low concentrations,28 remained growth inhibitory for the DTHMZ cells at all concentrations tested (0.1, 1 and 5
The Met proto-oncogene In addition to MyoD1, the introduction of the met proto-oncogene has also been described to induce transdifferentiation.29 The met proto-oncogene, a tyrosine kinase growth factor receptor30 which binds hepatocyte growth factor, mediates liver regeneration in vivo31 and allows MDCK epithelial cells to differentiate into branching tubules.32 When the met protooncogene was introduced into mouse NIH 3T3 fibroblasts they formed tumors which showed a lumen-like morphology in focal carcinoma-like areas containing typical epithelial cell junctions (desmosomes) and intermediate filaments (keratins). The question as to whether this transdifferentiation from mesenchymal to epithelial was complete and whether it was induced in a one-step process by the met protooncogene itself remains unclear at present. However, this finding provides further evidence that transdifferentiation processes from one embryonic lineage to another can be induced experimentally and may also be of relevance for developmental processes in the intact organism.
Transdifferentiation in intact organisms Finally, Kuziora and McGinnis33 demonstrated that transdifferentiation is not only a tissue culture phenomenon but can also be experimentally induced in the intact organism. They raised a fly stock in which 160
Gene transfer
Figure 2. Histological sections of 4-wk-old transplants. The cells are transplanted as intact cultures on a collagen matrix (col) onto the muscle fascia (mf) of nude mice. (A) while HaCaT cells develop into a regularly stratified and well differentiated epidermis (B) 5-aza-CR treated MyoD1 transfectants form a morphologically undifferentiated multi-layered tissue and (C) DTHMZ cells mostly a monolayer on top of the collagen gel. Bar, 25 µm.
161
P. Boukamp endogenous differentiation-specific genes are required to cooperate with the ‘foreign’ gene in order to induce and maintain the transdifferentiated phenotype. Thus, in analogy to the identification of genes of the MyoD family, transdifferentiation experiments may not only provide a deeper insight into individual differentiation processes but may also help to identify determination genes for differentiation of tissues other than muscle.
Acknowledgement Figure 3. RT-PCR analysis using specific primers for G-CSF and GM-CSF show a good expression in human fibroblasts (c and f) while HaCaT (a and d) and similarly DTHMZ cells (b and e) remain negative. The marker (M) used was QX174-HaeIII
The author wishes to thank Drs M.M. Muller ¨ and A. Hulsen ¨ who contributed to some of the experiments described and B. Plagens and Dr. A. Borchert for critical reading of the manuscript. This work was in part supported by the German-American Cooperation Program.
the deformed (dfr) gene, which is required to define normal head development by specifying the mandibular and maxillary segments, was introduced in all cells. Induction of ectopic expression of the gene, which was under inducible control of a heat-shock promoter (Hsp 70), resulted in partial transformation of many of the head and thoracic segments toward a maxillary segment identity. Similarly, Gibson and Gehring34 reported on ectopic expression of the Antennapedia gene, also under the control of the heatshock (Hsp 70) promoter. They demonstrated that, depending on the stage of development, overexpression of this ectopic gene caused an antenna-toleg transformation. Thus, even in such a complex system as the intact organism with all its regulatory components, expression of a determinator gene can be effective and is able to induce a differentiation program otherwise suppressed in this environment.
References 1. Blau HM, Chiu C-P, Webster C (1983) Cytoplasmic activation of human nuclear genes in stable heterocaryons. Cell 32:1171-1180. 2. Blau HM, Pavlath GK, Hardeman EC, Chiu CP, Silberstein L, Miller SG, Webster C (1985) Plasticity of the differentiated state. Science 230:758-766. 3. Hardeman E, Chiu C-P, Minty A, Blau HM (1986) The pattern of actin expression in human fibroblasts X mouse muscle herterokaryons suggests that human muscle regulatory factors are produced. Cell 47:123-130. 4. Baron MH, Maniatis T (1986) Rapid reprogramming of globin gene expression in transient heterokaryons. Cell 46:591-602 5. Vesely J, Cihak A (1978) 5-azacytidine: mechanism of action and biological effects in mammalian cells. Pharmacol Ther 2:813-840 6. Jones PA, Taylor SM (1980) Cellular differentiation, cytidine analogs and DNA methylation. Cell 20:85-93 7. Taylor SM, Jones PA (1979) Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine. Cell 17:771-779 8. Konieczny SF, Emerson CP (1984) 5-azacytidine induction of stable mesodermal stem cell lineages from 10T1/2 cells: evidence for regulatory genes controlling determination. Cell 38:791-800 9. Darmon M, Nicolas J-F, Lamblin D (1984) 5-azacytidine is able to induce the conversion of teratocarcinoma-derived mesenchymal cells into epithelial cells. EMBO J 3:961-967 10. Semat A, Duprey P, Vasseur M, Darmon M (1986) Mesenchymal-epithelial conversion induced by 5-azacytidine: appearance of cytokeratin Endo-A messenger RNA. Differentiation 31:61-66 11. Lassar AB, Paterson BM, Weintraub H (1986) Transfection of a DNA locus that mediates the conversion of 10T1/2 fibroblasts to myoblasts. Cell 47:649-656 12. Davis RL, Weintraub H, Lassar AB (1987) Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51:987-1000
Summary As demonstrated by several examples, transdifferentiation can be experimentally induced following transfer of genes required for a different differentiation pathway. However, such studies only seem to be successful with cells capable of differentiation; e.g. HeLa cells which have lost their differentiation capacity were refractory to myogenic differentiation both in heterokaryons and following transfection of MyoD1.2,20 They could, however, be reactivated by 5-aza-CR treatment.35 This might indicate that a set of 162
Gene transfer 13. Braun T, Buschhausen-Denker G, Bober E, Tannich E, Arnold HH (1989) A novel human muscle factor related to but distinct from MyoD1 induces myogenic conversion in 10T1/2 fibroblasts. EMBO J 8:701-709 14. Pinney DF, Pearson-White SH, Konieczny SF, Latham KE, Emerson Jr CP (1988) Myogenic lineage determination and differentiation: evidence for a regulatory gene pathway. Cell 53:781-793 15. Wright WE, Sasoon DA, Lin VK (1989) Myogenin, a factor regulating myogenesis, has a domain homologous to MyoD. Cell 56:607-617 16. Trapscott SJ, Davis RL, Thayer MJ, Cheng P-F, Weintraub H, Lassar AB (1988) MyoD1: a nuclear phosphoprotein requiring a myc homology region to convert fibroblasts to myoblasts. Science 242:405-411 17. Sassoon D, Wright WE, Lin V, Lassar AB, Weintraub H, Buckingham M (1989) Expression of two myogenic regulatory factors, myogenin and MyoD1, during mouse embyrogenesis. Nature 341:303-307 18. Hiti AL, Bogenmann E, Gonzales F, Jones PA (1989) Expression of the MyoD1 muscle determination gene defines differentiation capability but not tumorigenicity of human rhabdomyosarcomas. Mol Cell Biol 9:4722-4730 19. Choi J, Costa ML, Mermelstein CS, Chagas C, Holtzer S, Holtzer H (1990) MyoD converts primary dermal fibroblasts, chondroblasts, smooth muscle, and retinal pigmented epithelial cells into striated mononucleated myoblasts and multinucleated myotubes. Proc Natl Acad Sci USA 87:7988-7992 20. Weintraub H, Tapscott SJ, Davis RL, Thayer MJ, Adam MA, Lassar AB, Miller AD (1989) Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. Proc Natl Acad Sci USA 86:5434-5438 21. Boukamp P, Petrusevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE (1988) Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 106:761-771 22. Boukamp P, Stanbridge EJ, Foo DY, Cerutti PA, Fusenig NE (1990a) c-Ha-ras oncogene expression in immortalized human keratinocytes (HaCaT) alters growth potential in vivo but lacks correlation with malignancy. Cancer Res 50:2840-2847 23. Boukamp P, Breitkreutz D, Stark H-J, Fusenig NE (1990b)
24.
25.
26.
27. 28. 29. 30. 31. 32. 33. 34. 35.
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Mesenchyme-mediated and endogeneous regulation of growth and differentiation of human skin keratinocytes derived from different body sites. Differentiation 44:150-161 Breitkreutz D, Boukamp P, Ryle CM, Stark H-J, Roop DR, Fusenig NE (1991) Epidermal morphogenesis and keratin expression in c-Ha-ras transfected tumorigenic clones of the human HaCaT cell line. Cancer Res 51:4402-4409 Boukamp P, Chen J, Gonzales F, Jones PA, Fusenig NE (1992) Progressive stages of ‘transdifferentiation’ from epidermal to mesenchymal phenotype induced by MyoD1 transfection, 5-aza2'-deoxycytidine treatment, and selection for reduced cell attachment in the human keratinocyte line HaCaT. J Cell Biol 116:1257-1271 Boukamp P, Fusenig NE (1993) ‘Trans-differentiation’ from epidermal to mesenchymal/myogenic phenotype is associated with a drastic change in cell-cell and cell-matrix adhesion molecules. J Cell Biol 120:981-993 Thayer MJ, Weintraub H (1990) Activation and repression of myogenesis in somatic cell hybrids: evidence for trans-negative regulation of MyoD in primary fibroblasts. Cell 63:23-32 Moses HL, Yang EY, Pietenpol JA (1990) TGF-β stimulation and inhibition of cell proliferation: a new mechanistic insight. Cell 63:245-247 Tsarfaty I, Rong S, Resau JH, Rulong S, da Silva PP, Vande Woude GF (1994) The Met proto-oncogene mesenchymal to epithelial conversion. Science 263:98-101 Park M (1987) Sequence of MET protooncogene cDNA has features characteristic of the tyrosine kinase family of growth factor receptors. Proc Natl Acad Sci USA 84:6379-6383 Michalopoulos GK (1990) Liver regeneration: molecular mechanisms of growth control. FASEB J 4:176-187 Montesano R, Schaller G, Orci L (1991) Induction of epithelial tubular morphogenesis in vitro by fibroblast soluble factors. Cell 66:697-711 Kuziora MA, McGinnis W (1988) Autoregulation of a drosophila homeotic selector gene. Cell 55:477-485 Gibson G, Gehring WJ (1988) Head and thoracic transformation caused by ectopic expression of antennapedia during drosophila development. Development 102:657-675 Chiu C-P, Blau HM (1985) 5-azacytidine permits gene activation in a previously noninducible cell type. Cell 40:417-424
CELL BIOLOGY, Vol 6, 1995: pp 157–163
Transdifferentiation induced by gene transfer Petra Boukamp
and which by itself is able to induce transdifferentiation even across developmental cell lineage borders.
While for many tissues the differentiation process is well characterized, little is known about ‘master switch’ genes determining a specific differentiation pathway and having the potential to induce this process in a cell designed for a different differentiation pathway. Based on heterokaryon and 5-aza-cytidine-induced hypomethylation experiments, the muscle determination gene MyoD1 was identified and isolated, which was shown to induce myogenic differentiation even in cells of ectodermal lineage. Since transdifferentiation studies could also be performed in drosophila in vivo by ‘false’ expression of developmental genes, it is tempting to speculate that experimentally induced transdifferentiation mimics processes during embryonic development and tissue maturation.
Transdifferentiation by somatic cell fusion First evidence for the existence of transacting factors able to either repress or induce the expression of differentiated functions derived from somatic cell hybrid studies in the early 70s, when two cell types of different origin were fused with each other (see ref 1). However, due to frequent loss of chromosomes in these somatic cell hybrids, they proved to be rather unstable and highly variable model systems. Based on this, Blau and coworkers developed a heterokaryon system in which the intact nuclei of both fused cell types were stably maintained for the length of the experiments. Using representatives of all three embryonic lineages, heterokaryon experiments for the first time allowed to address the question as to what extent the origin of one fusion partner would influence the expression of the other along a different differentiation pathway. By fusion of mouse muscle cells with human amniotic fibroblasts, it was possible to show that a set of human muscle genes was activated including those coding for proteins of the contractile apparatus, membrane components and muscle specific enzymes.1,2 Furthermore, these genes were not induced randomly but followed the timecourse of human myogenesis.3 In this context, it is interesting to note that muscle gene activation was generally seen when primary (normal) human nonmuscle cells were fused with the mouse muscle cells, although the extent varied and muscle genes were expressed more rapidly and to a higher degree in more closely related cell types (fibroblasts [mesoderm] versus keratinocytes [ectoderm] or hepatocytes [endoderm]. Transformed aneuploid cells, on the other hand, often failed to express muscle markers. These primary findings were not only substantiated by others using similar cell types, but could also be extended to heterokaryons, where adult mouse erythroleukemia cells were fused with either human fetal erythroid or other non-erythroid cells and monitored for activation of the adult form of the globin gene.4
Key words: somatic-cell-fusion / 5-aza cytidine / epidermal differentiation / MyoD1-transfection
TOTIPOTENT CELLS DEVELOP into three distinct cell lineages in the very early embryo — ectoderm, endoderm and mesoderm. Once a cell is determined, this cell type is bound to specialize along a specific differentiation pathway, i.e. to develop into tissues such as muscle or epidermis and this determination is stably inherited by all daughter cells. From the characterization of the different differentiation pathways we have learned a lot about structural genes, membrane components involved in cell–cell and cell–matrix interactions, or enzymes that are specifically expressed and are characteristic for a given tissue type. These studies, however, did not lead to the identification of genes, so called determination genes, which control the concerted action of a differentiation-specific pathway. The first and at present still only known tissue determinator is the myogenic determination gene MyoD1, which was identified through ‘transdifferentiation’ studies where cells developed into an unrelated phenotype by ectopically forced expression of otherwise silent genes
From the German Cancer Research Center, Department of Carcinogenesis and Differentiation, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany ©1995 Academic Press Ltd 1043-4682/95/030157 + 07$8.00/0
157
P. Boukamp sional gel-electrophoresis studies indicated that only a few specific genes could be involved in lineage determination and that regulatory loci responsible for the myogenic determination, rather than differentiation-specific structural genes, were activated by 5-azaCR.8 Similarly, in mouse teratocarcinoma-derived fibroblasts, hypomethylation by 5-aza-CR resulted in a transient expression of a 55 kd keratin protein (K14), an intermediate filament protein characteristic for epithelial cells and generally absent in fibroblasts. From these cells, clones with epithelial morphology could be established, which not only expressed this keratin but, in addition, the whole set of epidermal keratins and desmosomes. Furthermore, these cells were able to differentiate into corneocytes.9,10 Thus, several authors were able to show that by treating cells with 5-aza-CR, genes were activated which then induced an alternative differentiation pathway; i.e. induced transdifferentiation. Moreover, since as an intriguing feature this cytidine analog selectively activates genes instead of causing genomewide random derepression this approach closely resembles the experimental design of transfer of specific genes. Thus, it was not surprising that the 5-aza-CR treated C3H 10T1/2 clone 8 cells were further used as a model system to determine genetic factors controlling the different mesodermal cell lineage determination and differentiation.
Thus, it was suggested from these heterokaryon experiments (i) that alternative differentiation pathways were not irreversibly blocked in already determined somatic cells and (ii) since myogenesis could be induced in the nucleus of the fusion partner this alternative differentiation pathway was not silenced by inactivating all the individual differentiation markers but only one or a few transacting factors.
Transdifferentiation by 5-azacytidine treatment This first step of ‘transdifferentiation’ could be extended to an even more completely transdifferentiated phenotype from fibroblastic to myogenic by treating C3H 1OT1/2 cells with 5-azacytidine (5-azaCR). Although it does not represent a true gene transfer, these experiments were based on the activation of genes which under normal conditions were never expressed in these cells. Furthermore, it was only because of this experimental approach that the determination gene for myogenesis was identified and that the present gene transfer experiments became feasible. 5-aza-CR, originally developed as a cancer chemotherapeutic agent,5 is a cytidine analog carrying a nitrogen instead of a carbon atom at position 5 of the pyrimidine ring and is randomly incorporated into DNA. Since the azanucleoside ring cannot be methylated, this integration now leads to extensive hypomethylation in CpG residues via inhibition of DNA methylation.6 By treating C3H 10T1/2 clone 8 cells with 5-aza-CR these multipotent mesodermal cells could be induced to develop into three new mesenchymal phenotypes. They formed contractile striated muscle cells which expressed elevated levels of myosin ATPase activity and acetylcholine receptors, biochemically defined adipocytes which accumulated large lipid droplets, and chrondrocytes which were able to synthesize cartilage-specific proteins.7 These altered phenotypes were not due to selection of rare pre-existing subpopulations since a comparable conversion never occurred spontaneously, and they were shown to be inheritable by propagation of the cells in the absence of further drug treatment. From these studies it was suggested that regulation of the three differentiation pathways had been inactivated in the precursor cell but could be reactivated by hypomethylation of specific genes. It remained unclear from these early studies whether the whole set or only one or a few ‘master switch’ genes had to be activated by hypomethylation. Subsequent two-dimen-
Transdifferentiation by gene transfer Genomic DNA The hypothesis that the profound effects of 5-aza-CR on differentiation/transactivation was due to the activation of one or a few determination loci (‘master switch’ genes) was substantiated by exciting experiments performed by Lassar and coworkers.11 They were able to show that DNA from 5-aza-CR-derived myoblasts, when transfected into normal (untreated) C3H 10T1/2 cells, induced the emergence of myoblasts at a frequency expected when only a few demethylated loci were required.
The master regulatory gene for myogenesis MyoD1 In an attempt to identify the genes, Lassar and coworkers used the technique of subtractive cDNA hybridization using poly(A) + RNA from normal diploid mouse myogenic cells and the 5-aza-CR treated 158
Gene transfer 10T1/2 cells.12 By screening a myocyte cDNA library a number of cDNA clones could be isolated, one of which was called MyoD1 and was sufficient to convert 10T1/2 cells, and, to a lesser extent, mouse 3T3 fibroblasts and a variety of other cell lines into stable myoblasts upon transfection. Furthermore, MyoD1 could be shown to be normally expressed in mouse skeletal muscle in vivo and in myogenic cell lines in vitro but to be absent in other tested adult or newborn mouse tissues as well as in several non-differentiating myogenic cell lines.12 Concurrently, other MyoD-like genes were identified which could similarly convert 10T1/2 cells into myogenic lineages.13-15 Once the MyoD1 gene was isolated, a number of studies were performed to demonstrate its potential to convert different fibroblast, adipoblast and osteosarcoma cell lines16-18 into muscle cells. In addition, it was asked whether this single ‘master switch’ gene MyoD1 would be able to also convert cells from unrelated embryonic lineages to myogenesis, i.e. to express muscle markers. And indeed it could be shown that transfection of retinal pigmented cells, mouse melanoma cells and rat neuroblastoma cells with this single myogenic determination gene resulted in the formation of multinucleated myotubes and coexpression of the endogenous and myogenic differentiation program.19,20 We have performed an extensive study using cells of ectodermal lineage, the human skin keratinocyte line HaCaT.21 The rational for this study was that epidermal differentiation in its tissue-specific organization is extremely well defined and that experimental systems are available to study this complex system. Furthermore, the HaCaT cells, although immortal and aneuploid, had maintained a largely normal capacity for epidermal differentiation even during serial passaging, or when the cells became malignantly transformed, and remained capable to form an epidermis-like epithelium under physiological conditions in vivo (in transplants) which closely resembled that of normal human keratinocytes.22-24 Thus, the HaCaT cells provided an excellent model system to study short- and long-term effects following stable integration of the MyoD1 gene and further propagation of individual clones.25 As expected, a small fraction of HaCaT cells converted into multinucleated myotubes (Figure 1A) and expressed the typical structural proteins vimentin, desmin (Figure 1B) and myosin. However, when the cells were grown in transplants in vivo and were exposed to the influence of the connective tissue (known to be required for complete epidermal differentiation) in addition to its
Figure 1. (A) Culture of MyoD1 transfected HaCaT cells. On a background of typical epidermal cells multinucleated myotubes have formed which are stained with an antibody against myosin. (B) Immunofluorescence micrograph of a similar culture using an anti-desmin antibody. (C) Phase contrast micrograph of DTHMZ cells demonstrating a more fibroblastic morphology. Bar, 100µm
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P. Boukamp ng/ml; Boukamp and H¨ulsen, unpublished results). Furthermore, expression of growth factors characteristic for fibroblasts such as granulocyte- or granulocyte/macrophage-colony-stimulatory factor (G-CSF or GM-CSF) was similarly absent in HaCaT as in DTHMZ cells (Boukamp and M¨uller, unpublished results). Thus, these studies strongly suggest that there are independent regulatory pathways for (i) the expression of and reactivity to growth factors and (ii) ‘morphological’ differentiation. We further have to postulate that already for ‘morphological’ transdifferentiation from ectodermal to mesenchymal lineage one ‘master switch gene’ is not sufficient to overcome the stable intrinsic program (at least for epidermal differentiation), since variants similar to DTHMZ cells did not evolve from HaCaT or MyoD1- transfectants without 5-aza-CR treatment. Only by additional hypomethylation we obviously activated one or more of the genes required to induce this fully transdifferentiated phenotype.
intrinsic regulation, the myogenic differentiation pathway was down-regulated and the cells formed a normally structured and biochemically well-differentiated epidermis.25 (Figure 2A). From these MyoD1 transfectants we were able, in a step-wise fashion, to select for a variant with solely mesenchymal/myogenic characteristics. To potentiate the MyoD1 action, MyoD1-transfectants were treated with 5-aza-CR. Although this did not result in an increase in the number of myogenic cells in vitro, it clearly altered the response to intrinsic/extrinsic regulators of epidermal differentiation in vivo.25 The epithelium formed by these cells was morphologically unstructured and undifferentiated (Figure 2B). Nevertheless, differentiation markers were still expressed in a rather normal fashion. Since a similar dissociation of histo- and cytodifferentiation is seen during embryological development when the epidermis develops from a two- to a multi-layered tissue, the experimentally induced stage may represent the physiological ‘transition-phase’ and thus, the ModD1transfected, 5-aza-CR-treated HaCaT cells may provide a useful model to determine factors involved in epidermal maturation and differentiation. In these cultures a subpopulation (DTHMZ) evolved which, when selected on the basis of reduced adhesion, had reached a stage of ‘complete’ transdifferentiation25 (Figure 1C). Reduced adhesion could clearly be related to the loss of expression in a group of adhesion molecules, the integrins, which were transformed from a complex keratinocyte-like pattern to a more simple fibroblast-like one.26 As a functional consequence, growth on those matrices was poor where the respective receptors were missing. But even on growth-promoting substrates (fibronectin) the DTHMZ cells were no longer able to stratify when grown as transplants in vivo. They mostly remained as a monolayer (Figure 2C) and no longer expressed any of the epidermal differentiation markers either in vivo or in vitro. Instead, the number of cells expressing myogenic differentiation markers doubled and, additionally, other myogenic regulator genes such as myf-313 and myogenin shown to be activated in a positive autoregulatory loop with MyoD1,27 were seen. Despite of all these alterations, the functional switch to mesenchymal/myogenic phenotype was still not complete. Recent experiments have shown that TGF-β1, a potent growth-inhibitor for keratinocytes, which, however, stimulates growth of fibroblasts at low concentrations,28 remained growth inhibitory for the DTHMZ cells at all concentrations tested (0.1, 1 and 5
The Met proto-oncogene In addition to MyoD1, the introduction of the met proto-oncogene has also been described to induce transdifferentiation.29 The met proto-oncogene, a tyrosine kinase growth factor receptor30 which binds hepatocyte growth factor, mediates liver regeneration in vivo31 and allows MDCK epithelial cells to differentiate into branching tubules.32 When the met protooncogene was introduced into mouse NIH 3T3 fibroblasts they formed tumors which showed a lumen-like morphology in focal carcinoma-like areas containing typical epithelial cell junctions (desmosomes) and intermediate filaments (keratins). The question as to whether this transdifferentiation from mesenchymal to epithelial was complete and whether it was induced in a one-step process by the met protooncogene itself remains unclear at present. However, this finding provides further evidence that transdifferentiation processes from one embryonic lineage to another can be induced experimentally and may also be of relevance for developmental processes in the intact organism.
Transdifferentiation in intact organisms Finally, Kuziora and McGinnis33 demonstrated that transdifferentiation is not only a tissue culture phenomenon but can also be experimentally induced in the intact organism. They raised a fly stock in which 160
Gene transfer
Figure 2. Histological sections of 4-wk-old transplants. The cells are transplanted as intact cultures on a collagen matrix (col) onto the muscle fascia (mf) of nude mice. (A) while HaCaT cells develop into a regularly stratified and well differentiated epidermis (B) 5-aza-CR treated MyoD1 transfectants form a morphologically undifferentiated multi-layered tissue and (C) DTHMZ cells mostly a monolayer on top of the collagen gel. Bar, 25 µm.
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P. Boukamp endogenous differentiation-specific genes are required to cooperate with the ‘foreign’ gene in order to induce and maintain the transdifferentiated phenotype. Thus, in analogy to the identification of genes of the MyoD family, transdifferentiation experiments may not only provide a deeper insight into individual differentiation processes but may also help to identify determination genes for differentiation of tissues other than muscle.
Acknowledgement Figure 3. RT-PCR analysis using specific primers for G-CSF and GM-CSF show a good expression in human fibroblasts (c and f) while HaCaT (a and d) and similarly DTHMZ cells (b and e) remain negative. The marker (M) used was QX174-HaeIII
The author wishes to thank Drs M.M. Muller ¨ and A. Hulsen ¨ who contributed to some of the experiments described and B. Plagens and Dr. A. Borchert for critical reading of the manuscript. This work was in part supported by the German-American Cooperation Program.
the deformed (dfr) gene, which is required to define normal head development by specifying the mandibular and maxillary segments, was introduced in all cells. Induction of ectopic expression of the gene, which was under inducible control of a heat-shock promoter (Hsp 70), resulted in partial transformation of many of the head and thoracic segments toward a maxillary segment identity. Similarly, Gibson and Gehring34 reported on ectopic expression of the Antennapedia gene, also under the control of the heatshock (Hsp 70) promoter. They demonstrated that, depending on the stage of development, overexpression of this ectopic gene caused an antenna-toleg transformation. Thus, even in such a complex system as the intact organism with all its regulatory components, expression of a determinator gene can be effective and is able to induce a differentiation program otherwise suppressed in this environment.
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Summary As demonstrated by several examples, transdifferentiation can be experimentally induced following transfer of genes required for a different differentiation pathway. However, such studies only seem to be successful with cells capable of differentiation; e.g. HeLa cells which have lost their differentiation capacity were refractory to myogenic differentiation both in heterokaryons and following transfection of MyoD1.2,20 They could, however, be reactivated by 5-aza-CR treatment.35 This might indicate that a set of 162
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